Immune Mechanisms in Pain Control

Classically, pain sensation or suppression has been attributed exclusively to neuronal circuits. This review challenges this notion and presents an expanded concept about the contribution of immune mechanisms in the inhibition of pain (analgesia). Among the many transmitters with potential for neuroimmune interactions, we concentrate here on opioids, the most extensively investigated compounds. Pain inhibition mediated by immune-derived opioids results from interactions with the peripheral nervous system. A prerequisite for the manifestation of such effects seems to be inflammation, accompanied by hyperalgesia. Opioid receptors are present on peripheral terminals of sensory neurons and are upregulated in inflammation. Their endogenous ligands, opioid peptides, are synthesized in circulating immune cells that under pathological conditions migrate to injured tissue. This is orchestrated by selectins and other adhesion molecules located on opioid-containing immune cells and on vascular endothelium. Under stressful stimuli or in response to releasing agents (e.g., corticotropin-releasing hormone or cytokines), immunocytes can secrete opioids. These peptides activate peripheral opioid receptors and produce analgesia by inhibiting the excitability of sensory nerves, the release of excitatory neuropeptides, or both. Because these effects occur in the periphery, they are devoid of central opioid side effects, such as respiratory depression, sedation, dysphoria, or dependence. Targeting of immune cells containing opioids to injured tissues is a novel concept of pain control and opens potential new therapeutic approaches. Peripheral Opioid Receptors Localization All three types of opioid receptors (μ, δ, and κ) are expressed within sensory neurons. These receptors have been found on cell bodies in the dorsal root ganglia and on peripheral terminals of primary afferent neurons in animals (1–7) and in humans (8). Binding experiments indicate that the characteristics of peripheral opioid receptors are very similar to those in the brain (2). Furthermore, the molecular mass of the peripheral μ opioid receptor is identical with that in the spinal cord (6). In vivo experiments have confirmed the functional significance of these receptors in that the peripheral analgesic effects of μ-, δ-, and κ-selective agonists are abolished by pretreatment with capsaicin, a neurotoxin selective for primary afferent C-fibers (9). It has been suggested that opioid receptors are also located on sympathetic postganglionic neuron terminals. However, there are reports refuting this notion, and studies attempting the direct demonstration of opioid receptor messenger RNA (mRNA) in sympathetic ganglia have produced negative results (reviewed in Ref. 10). In addition, thorough morphological investigations have clearly demonstrated the presence of δ opioid receptors on unmyelinated primary afferent neurons and the absence of such receptors on postganglionic sympathetic neurons in skin, lip, and cornea (11). Moreover, chemical sympathectomy with 6-hydroxydopamine does not change the expression of opioid receptors in the dorsal root ganglion or the peripheral analgesic effects of μ-, δ-, and κ-opioid agonists in a model of inflammatory pain (9,12). Together, these findings have corroborated the notion that peripheral opioid receptors mediating analgesia are exclusively localized on primary sensory neurons. Opioid binding sites and the expression of opioid receptor transcripts have also been demonstrated in immune cells (2,13,14). Opioid-mediated modulation of the proliferation of these cells and of their functions (e.g., chemotaxis, superoxide and cytokine production, and mast cell degranulation) has been reported (15). These immunomodulatory actions can be stimulatory as well as inhibitory and have been ascribed to the activation of opioid receptors (13,15,16). However, the significance of such effects with regard to pain transmission has not been investigated. Alterations During Inflammation Opioid receptors are synthesized in the dorsal root ganglia (3,4,17). Axonal transport is responsible for delivering macromolecules from the cell body to nerve terminals. After the induction of peripheral inflammation, the axonal transport of opioid receptors in fibers of the sciatic nerve is greatly enhanced (2,3,6). Subsequently, the density of opioid receptors on cutaneous nerve fibers in the inflamed tissue increases, and this increase is abolished by ligation of the sciatic nerve (2,6). These findings indicate that inflammation enhances the peripherally directed axonal transport of opioid receptors, which leads to an increase in their number (upregulation) on peripheral sensory nerve terminals. Also, preexistent, but possibly inactive, neuronal opioid receptors may undergo changes owing to the specific milieu (e.g., low pH value) of inflamed tissue and thus be rendered active. Indeed, low pH values increase opioid agonist efficacy in vitro by altering the interaction of opioid receptors with G-proteins in neuronal membranes (18). Furthermore, inflammation entails a disruption of the perineurium that normally acts as a diffusion barrier for high molecular weight or hydrophilic substances such as peptides (19). In addition, the number of primary afferent terminals is increased in the inflamed tissue (sprouting) (6). These observations suggest that the access of opioid ligands to opioid receptors on primary afferent neurons is greatly facilitated in inflamed tissues. Neuronal Mechanisms Opioids produce analgesia by several mechanisms. In cells of the central nervous system (CNS), they increase potassium and decrease calcium currents through interactions with G-proteins (Gi and Go) (20). In dorsal root ganglion neurons, opioids can inhibit calcium currents (21). Their effect on potassium currents is a matter of controversy. When calcium channels were not blocked, opioids were found to decrease or increase potassium currents depending on their concentrations (22) or to inhibit calcium-dependent potassium currents (21). However, in the presence of calcium channel blockers, opioid effects on resting or voltage-dependent potassium channels could not be detected (21). Finally, the inhibition of a tetrodotoxin-resistant sodium current by a μ opioid agonist was described. Thus, opioids can attenuate the excitability of the peripheral terminals of sensory neurons and the propagation of action potentials. Also, they can inhibit the (calcium-dependent) release of excitatory neurotransmitters (e.g., substance P) from peripheral sensory nerve endings (reviewed in Refs. 10,23). Analgesic Effects of Exogenous Opioids The notion of analgesia mediated by immune-derived opioids was preceded by the demonstration of peripheral analgesia produced by exogenous opioids. These effects were initially observed after administration of opioid agonists in small, systemically inactive doses directly into injured tissue in models of neuropathic pain, colorectal distension, bone damage, and inflammation, also in noninflamed tissue (reviewed in Refs. 24,25). Central side effects are avoided by this peripheral application of drugs. Subsequently, strategies to restrict the access of opioid agonists to the CNS have been developed. The goal is to achieve peripheral selectivity and high analgesic potency of compounds that are unable to cross the blood-brain barrier (reviewed in Refs. 23,26,27). Recently, for example, novel peptidic peripherally selective κ-ligands exhibiting potent analgesic and antiinflammatory effects were developed (28). Such peripheral opioid actions are of definite clinical relevance. A sizeable body of literature has demonstrated the analgesic efficacy of locally applied opioids in various clinical settings, including the intraabdominal, orbital, or topical wound infiltration, and the intraarticular application after knee surgery or in chronic arthritis (26,29,30). These studies have clearly demonstrated for the clinical usefulness of peripherally active opioid analgesics. Opioid Peptides Produced by Immune Cells Opioid peptides are the natural ligands at opioid receptors. Three families of these peptides are well characterized in the CNS and neuroendocrine system. Each family derives from a distinct gene and from one of the three precursor proteins proopiomelanocortin (POMC), proenkephalin (PENK), or prodynorphin. Appropriate processing yields their respective representative opioid peptides, the endorphins, enkephalins, and dynorphins. These peptides exhibit different affinity and selectivity for the three opioid receptors μ (endorphins and enkephalins), δ (enkephalins and endorphins), and κ (dynorphin) (31). Two additional endogenous opioid peptides have been isolated from the bovine brain—endomorphin-1 and endomorphin-2. Both peptides are considered highly selective μ receptor ligands (32). Their precursors are not yet known. All of these opioid peptides have been detected in immune cells, but the POMC and PENK families have been studied most extensively. POMC-Derived Opioid Peptides POMC-derived peptides in immune cells are well conserved during evolution (33). Blalock and Smith were the first to demonstrate POMC expression by leukocytes at the protein level (34,35). Since then, POMC-related opioid peptides have been found in immune cells of many vertebrates and nonvertebrates (33–37). To determine whether these immune-competent cells actually synthesize POMC rather than simply absorb related peptides from plasma, mRNA encoding POMC was sought for and demonstrated in many of these studies (35). The pituitary POMC gene is organized into three exons separated by intervening sequences that are removed during processing after transcription to produce the full length 1200 nt transcript (31). Initially, truncated POMC transcripts were found in leukocytes (38–40). Lyons and Blalock (41) re-examined the question of POMC mRNA expression using novel polymerase chain reaction procedures. With this exacting and sensitive methodology, they found expression of full-length transcripts encoding all three POMC exons in rat mononuclear leukocytes. This POMC transcript is spliced in the same way as the pituitary transcript and, consequently, contains the sequence for the signal peptide required for the correct routing into the secretory pathway. The POMC protein is also proteolytically processed in a way consistent with the pituitary gland (41). These results unequivocally demonstrate that immune cells can produce full-length POMC transcripts. Apparently, this production is stimulated by various immune and inflammatory mediators (35). PENK-Derived Opioid Peptides PENK-derived opioid peptides have also been detected in human and rodent immune cells (35,42,43). Both the mRNA and met-enkephalin protein were detected. Preproenkephalin mRNA was found in T- and B-cells, macrophages, and mast cells (44). In subpopulations of immune cells, this mRNA is highly homologous to brain PENK mRNA, abundant, and apparently translated because immunoreactive enkephalin is present, released, or both (reviewed in Ref. 42). The appropriate enzymes required for posttranslational processing of both POMC and PENK have also been identified in immune cells (45). Immune-Derived Opioid Peptides in Inflammation Immune-derived opioid peptides apparently play a substantial role in the modulation of inflammatory pain (46,47). Persistent inflammation is a pathophysiological in vivo stimulus for the immune system and represents a condition that is closer to the clinical setting than in vitro studies. POMC mRNA and β-endorphin, as well as met-enkephalin and dynorphin, were found in circulating lymphocytes and in cells derived from lymph nodes of rats with and without localized hind paw inflammation (48,49). In the same model, mRNAs encoding POMC and PENK and the corresponding opioid peptides β-endorphin and met-enkephalin are abundant in cells of inflamed but not in noninflamed tissue (1,48–50). Small amounts of dynorphin are also detectable by immunohistochemistry (51). Histomorphological procedures and flow cytometry have identified the opioid-containing cells as T- and B-lymphocytes, granulocytes, and monocytes/macrophages (50–52). Recent studies using double immunofluorescence produced additional anatomical evidence that β-endorphin is present in activated/memory T cells within inflamed tissue (6,49). Thus, opioid peptides are processed and present both in circulating and resident inflammatory cells at the site of injury. Other peripheral loci of opioid peptide production include the adrenals, pituitary, or primary afferent neurons. However, these were excluded as functionally relevant sources for the generation of opioid analgesia (46,47). Migration of Opioid-Containing Immune Cells to Inflamed Tissue The recruitment of leukocytes from circulation into areas of inflammation begins with the attachment of these cells to vascular endothelium, followed by their transmigration into the tissue. Although this observation has been documented for more than 150 years, only the last decade uncovered the molecular mechanisms underlying leukocyte extravasation with the identification of specific cell adhesion and chemoattractant/activator molecules. Leukocytes are recruited from the circulation by a well-orchestrated set of events. They undergo multiple attachments to and detachments from the vessel’s endothelial cells before transendothelial migration. This includes slowing and rolling along the endothelial cell wall that is mediated predominantly by the interaction of selectins expressed on leukocytes (L-selectin) and on endothelial cells (P- and E-selectin) with their ligands on endothelium or immune cells, respectively. The rolling immunocytes can then be activated by chemoattractants released from inflammatory cells and endothelium. This leads to the upregulation and increased avidity of integrins. These mediate the firm adhesion of leukocytes to endothelial cells via ligands of the immunoglobulin superfamily. Finally, the immune cells transmigrate through the endothelial wall mediated by immunoglobulin superfamily members (e.g., platelet-endothelial adhesion molecule-1) and are directed to the sites of inflammation to initiate a host defense (reviewed in Refs. 53,54). Recent findings indicate that these events can also be involved in the endogenous control of inflammatory pain. We have shown that L-selectin is expressed on lymphocytes and macrophages in lymph nodes and on cells that have migrated to inflamed subcutaneous paw tissue in rats. P-selectin and platelet-endothelial adhesion molecule-1 are constitutively expressed on the endothelium of blood vessels in noninflamed lymph nodes and subcutaneous paw tissue, and they are upregulated in inflammation. More importantly, double immunofluorescence demonstrated that L-selectin is co-localized with β-endorphin in leukocytes in lymph nodes and in inflamed paw tissue (55). Furthermore, pretreatment of rats with a selectin blocker (fucoidin) decreases the number of β-endorphin–containing immunocytes infiltrating the inflamed tissue (56). In consequence, this diminishes the β-endorphin content in inflamed tissue and concurrently abolishes endogenous peripheral opioid analgesia (see “Endogenous Opioid Analgesia” below and Ref. 56). This suggests that circulating opioid-producing immunocytes migrate to inflamed tissue where they secrete the opioids to inhibit pain. Afterwards, they travel to the regional lymph nodes depleted of the opioid peptides (48,49). Thus, local signals apparently not only stimulate the synthesis of opioid peptides in resident inflammatory cells, but also attract opioid-containing cells from the circulation to the site of injury to reduce pain. This is controlled by specific adhesive mechanisms. Release of Opioid Peptides from Immune Cells In the pituitary, β-endorphin and other POMC-derived peptides are released by corticotropin-releasing hormone (CRH) and interleukin-1β (IL-1) (reviewed in Ref. 57). Similar mechanisms can trigger opioid release within peripheral inflamed tissue. CRH is present in immune cells, fibroblasts, and vascular endothelium. Interestingly, peripheral CRH expression is enhanced in inflamed synovial and subcutaneous tissue of animals and humans (reviewed in Ref. 57). In a rat model of paw inflammation, autoradiography revealed CRH and IL-1 receptors and their upregulation in inflamed lymph nodes and paw tissue. They were located on lymphocytes and macrophages but not on peripheral sensory nerves. Their pharmacological characteristics were similar to the high-affinity CRH and IL-1 binding sites in the pituitary (58). In line with these studies, it was shown that lipopolysaccharide stimulation resulted in a dramatic increase of CRH receptors on neutrophils, granulocytes, and macrophages in the mouse spleen (59). Activation of CRH and IL-1 receptors on cells from inflamed lymph nodes results in the secretion of opioid peptides (48,49,60). In those studies, β-endorphin, met-enkephalin, and dynorphin were dose-dependently released by CRH, whereas IL-1 released β-endorphin and dynorphin but not met-enkephalin. These effects were dose-dependently antagonized by the respective CRH and IL-1 receptor antagonists, indicating a specific effect mediated by CRH and IL-1 receptors (48,49,60). Moreover, this release of opioid peptides was calcium-dependent and mimicked by increased extracellular concentrations of potassium. This is consistent with a regulated pathway of release from secretory vesicles, as in neurons and endocrine cells (48,49). Taken together, CRH and IL-1 act on their respective receptors on immune cells to elicit a release of opioid peptides from immune cells in vitro. Analgesia Produced by Immune-Derived Opioid Peptides Analgesic Effects of CRH and IL-1 Because CRH and IL-1 induce the release of opioids from immune cells in vitro, this might also occur in vivo. Indeed, both CRH and IL-1 injected directly into the inflamed paw produce dose-dependent analgesia reversible by their respective antagonists. Intravenous administration of these agents does not change pain thresholds, demonstrating a peripheral site of action (60). These results are in line with other studies on local analgesic effects of CRH (56,61) but are in contrast with reported IL-1-induced hyperalgesia (62). Importantly, however, in the latter study, IL-1 was injected into noninflamed tissue devoid of opioid containing cells. Apparently, such cells are the targets for CRH and IL-1 because immunosuppression with cyclosporine A as well as antiselectin treatment results in a significant reduction of opioid-containing cells and of CRH- and IL-1-induced analgesia (56,60). Furthermore, CRH- and IL-1-induced analgesia is blocked by an antibody β-endorphin, that this opioid a In addition, met-enkephalin seems to be involved in the CRH- and dynorphin in the IL-1-induced analgesia (60). These opioid peptides activate their receptors on sensory neurons to of inflammatory pain (60). is evidence that other immune-derived and can also decrease inflammatory pain. When injected directly into an inflamed tissue, these whereas their hyperalgesia by and This enhanced inflammatory pain by and not occur in mast or respectively. Thus, it seems that endogenous sources of and are mast cells and respectively. Their analgesic effects are attributed to the inhibition of the production or release of other such as and which (reviewed in Ref. Opioid Analgesia is the of CRH- and In by a potent analgesia in inflamed but not in noninflamed of inflammation this effect is mediated by peripheral μ and δ receptors A opioid peptide involved is β-endorphin because this analgesia is abolished by β-endorphin but not met-enkephalin or dynorphin Thus, analgesia in inflamed tissue can be through the activation of local opioid receptors by endogenous opioid peptides released during of this release include locally produced CRH and derived from sympathetic nerve endings immune cells are the of opioids is demonstrated by the of analgesia by immunosuppression with cyclosporine A or body Moreover, this effect is also by the extravasation of immune cells from of and (56). the number of cells and the of β-endorphin in the inflamed tissue are (56). Together, these findings demonstrate that and the of immune cells and the generation of pain control in injured tissue. Recently, the of subpopulations of opioid-containing immunocytes and their contribution to endogenous analgesia was in to the of inflammation In inflammation, a of opioid-producing leukocytes are granulocytes, whereas at and macrophages play a With of inflammation, the number of opioid-containing immunocytes and the β-endorphin content In peripheral analgesia Thus, the potency of endogenous pain inhibition is to the number of opioid cells, and distinct leukocyte to this at different of inflammation. Peripheral endogenous opioid analgesia is of clinical relevance. Opioid receptors are present on peripheral terminals of sensory in human and these receptors are of mediating analgesia in humans Opioid peptides are found in human synovial cells, mast cells, and The peptides are β-endorphin and met-enkephalin, whereas only amounts of dynorphin are detectable (8). The interaction of synovial opioids with peripheral opioid receptors was in knee intraarticular opioid receptors by the local administration of resulted in increased pain Taken together, these findings suggest that in a stressful (e.g., opioids are released from inflamed tissue and activate peripheral opioid receptors to attenuate clinical pain. Importantly, these endogenous opioids not with exogenous intraarticular is an potent analgesic in with and without opioid-producing inflammatory synovial cells (8). This suggests in contrast to the of in the the immune opioids not produce to at peripheral opioid receptors. control of inflammatory pain can from interactions the nervous and immune Immune cells opioid peptides migrate to the inflamed tissue. 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In response to or releasing such as corticotropin-releasing hormone (CRH) or interleukin-1β leukocytes secrete opioid CRH and IL-1 release opioids by with CRH receptors and IL-1 receptors respectively. Opioid peptides or exogenous opioids can to peripheral opioid receptors to include